Ribozymes John J. Rossi Beckman Research Institute of the City of Hope, Duarte, California, USA RNA enzymes or ribozymes are receiving considerable attention for their potential use as highly specific inhibitors of gene expression. From the basic science perspective, the mechanisms by which ribozymes catalyze site-specific cleavage (and in some cases ligation) reactions provide exciting and active areas of scientific investigation. The most recent developments in our understanding of the molecular mechanisms of catalysis, as well as in vivo applications of ribozymes, are highlighted. Current Opinion in Biotechnology 1992, 3:3-7
Introduction RNA, besides being an informational molecule, can also possess enzymatic properties. Those RNAs having this property have been termed ribozymes. Since the initial discoveries of enzymatic RNAs by Cech and Altman and their colleagues [1,2], there have been numerous descriptions of ribozymes found throughout nature. Aside from their important roles in biological processes such as splicing and RNA modification, as well as the implications for macromolecular evolution, the fact that RNAs can be enzymatic has fostered a whole new technology of transacting, catalytic, antisense ribozymes. This new class of antisense agents allows specific targeting of any desired RNA, followed by cleavage and functional inactivation of that target. Certain of these molecules have truly catalytic properties in that they are not consumed or modified in the reaction, and can turn over multiple substrates. In this review I will discuss first the various catalytic motifs, then the past year's literature describing structural and kinetic analyses of ribozymes and, finally, the most recent papers describing the applications of ribozymes in vivo as catalytic antisense agents.
RNA catalytic motifs At present, there are four RNA catalytic motifs that are potentially useful for tran.~mediated cleavage of target RNAs (Fig. 1). Perhaps the most thoroughly studied is that of the self-splicing intervening sequence of Tetrah)~ mena thermophila, first characterized by Cech and colleagues [ 1 ]. Although this RNA enzyme possesses a rather complex structure, it has been engineered to cleave a variety of substrates, even including single-stranded DNA [3,4"]. RNAse P is a ubiquitous enzyme that processes the 5' leader sequences from pre-transfer RNAs and has an RNA component that has been shown to be the catalytic entity for the bacterial form of the enzyme [2]. Forster and Altman [5"] have characterized the substrate requirements for the E. coli enzyme, and have demonstrated that it can cleave a non-tRNA duplex provided
that the target is in a helix and that the antisense to the target RNA has a CCA sequence at its 3' terminus (Fig. lb). Thus, in principle, any RNA can be targeted as a substrate for RNAse P provided that the CCA-containing antisense RNA can be annealed to the target RNA. Two RNA catalytic motifs that originate from plant viroids and virusoids have received a great deal of attention for their use in antisense ribozymes. These are the 'hammerhead' [6] and 'hairpin' [7] cleavage domains, illustrated in Fig. 1 (c and d). Uhlenbeck [8] and Haseloff and Gerlach [9] demonstrated that the hammerhead catalytic motif could be incorporated into trans-acting antisense RNAs, thereby imparting enzymatic activity on these molecules. The hairpin catalytic domain was subsequently shown to be useful for incorporation into tran.~acting ribozymes as well [6]. One other ribozyme catalytic domain that has potential for inclusion into trans.acting ribozymes is that derived from the human hepatitis delta virus. The catalytic center of this ribozyme, like that of the self-splicing introns and RNAse P, is more complex than that of the hammerhead and is heavily dependent upon structure for its activity [10-12]. A number of investigators have recently reported their efforts to define the catalytic domain of this molecule [11-13]. Once this ribozyme is better defined, it should also find application for inclusion into catalytic antisense RNAs.
Structural and kinetic analyses of ribozyme-mediated catalysis A large number of studies have sought to provide a better understanding of the mechanism of ribozyme-mediated catalysis, as well as to provide an intelligent framework for designing more active ribozymes. Two groups have used an in vitro evolution approach to create Tetrahymena-type ribozymes with altered or enhanced catalytic properties [4.-,14-.]. These investigators took advantage of the fact that the Tetrahymena ribozyme has both cleavage and ligation activities, and set up in vitro systems for enriching and amplifying ribozyme variants
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with desired characteristics. In these studies, a randomized population of modified ribozymes was incubated with the desired, specific substrate molecule that is to be cleaved. Concommitant with the cleavage event, substrate sequences lying 3' to the site of cleavage become ligated to ~ e intron. The intron, with its covalently attached cleavage product is amplified by the polymerase chain reaction using primers complementary to the cleavage product and the 5' end of the intron. At least one of the primers contains the sequence specifying transcription from a bacteriophage promoter such as T7. The amplified DNA is transcribed, and the whole process repeated several times until the modified form of the ribozyme that is most active in cleaving the desired substrate is the major
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Fig. 1. Four different RNA catalytic motifs: (a) Tetrahymena self-splicing intervening sequence ribozyme; (b) Esch erichia coil RNAse P, comprising RNAse P proteins and M1 RNA to make up the holoenzyme; (c) hammerhead ribozyme; and (d) the tobacco ringspot virus (negative strand) hairpin ribozyme. The guide sequence for the Tetrahymena ribozyme is IGS for internal guide sequence, and the RNAse P guide sequence is EGS for external guide sequence. The IGS is part of the Tetrahymena core ribozyme and is its primary binding site for substrate. Specific complementary base-pairing interactions between this site and the substrate confer specificity to the ribozyme reaction. The EGS is an 'antisense' RNA with an appended ACCA at the 3' end, which is provided in trans. Complementary base pairing of the EGS with the target RNA results in a duplex substrate for RNAse P, by mimicking the CCA stem of the natural tRNA substrate molecules. The symbols N and N' denote no specificity for nucleotides at these positions, and roman numerals indicate regions required for ribozyme functions. All four ribozymes are depicted as they would interact with substrates 'in trans'. The substrates and the sites of cleavage are indicated.
amplified product. By using DNA substrates in place of RNA, Robertson and Joyce [4 °°] were able to generate variants that have single-stranded DNA cleavage capabilities. Using a somewhat different strategy, Green et at [14o*] began with a pool of mutant ribozymes in which the wild-type sequence was present at an extremely low level. By carrying out only three cycles of selection and amplification for enhanced ribozyme function, they were able to isolate the wild-type ribozyme as well as structural variants as active as the wild type. Using such m vitro selection schemes, it should be possible to isolate variant Tetrahymena-type ribozymes capable of acting on virtually any substrate of choice. Unfortunately, it has not yet been possible to apply such a directed evolution strategy
Ribozymes Rossi S to the hammerhead-type ribozymes. Recently, an in vitro selection scheme, similar in principal to those used for the Terahymena ribozyme intron, has been developed for the hairpin ribozyme, allowing selection for cleavage of novel substrates [15]. Cech and his colleagues [16,17.-] have used the Tetrah?~ mena ribozyme system to determine the general principles of ribozyme-substrate interactions. A particularly interesting observation involves a tertiary interaction between a 2' hydroxyl group at a particular position in the susbtrate with residues within the Tetrahymena intron ribozyme. This single interaction results in an increase in binding energy of 4.1 kcal m o l - 1 over that expected for simple RNA-RNA base pairing [17-.]. This increased binding energy greatly reduces the efficiency of product dissociation, thereby slowing the turnover rate of the ribozyme. Such tertiary interactions probably exist for other types of ribozyme-substrate interactions as well and it may thus be possible to increase catalytic turnover of ribozymes by altering or changing these interactions. Several key papers analyzing hammerhead-type ribozymes have helped to define the essential nucleotides and magnesium-binding sites within the catalytic center. Ruffner et al. [18..] have rigorously evaluated the contribution to catalytic efficiency of every nucleotide in the hammerhead structure. Their data clearly demonstrate that the catalytic efficiency can be grossly altered by several different single base substitutions within the highly conserved hammerhead domain. The choice of cleavage site is also an important consideration, with GUC being the kinetically most favorable site. Perreault et al. [19] substituted deoxyribonucleotides at various positions in the hammerhead structure to assess the role of the 2' hydroxyl group on the ribose moiety in the binding of magnesium cations. Three positions, two of which reside in the hammerhead and one that is adjacent to the site of cleavage, were found to be critical for magnesium binding. On the basis of these observations, a molecular model for hammerhead ribozyme mediated cleavage is presented, which involves magnesium binding at these three critical positions and at the scissile phosphate. Slim and Gait [20] substituted configurationally defined phosphorothioate-containing oligoribonucleotides at a single site in a hammerhead ribozyme cleavage site to investigate the importance of magnesium ion binding to the pro-R oxygen of the cleaved phosphodiester. Their findings provide further evidence that a magnesium ion is bound to the pro-R oxygen in the transition state of the cleavage reaction. In combination, these results support a model in which the role of the magnesium ion is primarily catalytic rather than structural. Pieken et aL [21] chemically synthesized hammerhead ribozymes with either 2' fluoro or 2' aminonucleotides. The incorporation of 2' fluorouridines, 2' fluorocytidines or 2' aminouridines did not appreciably decrease catalytic efficiency, but did markedly increase the resistance of these ribozymes to ribonuclease degradation, an important consideration for exogenous delivery of ribozymes to cells in culture. Each of the above studies has provided greater insight into the roles of the highly con-
served bases and ribose moieties in the catalytic center of the hammerhead ribozyme and at the site of cleavage of its substrates. Further physical studies, however, using nuclear magnetic resonance spectroscopy and X-ray crystallographic analyses will be necessary for a full understanding of the mechanism of the site-specific cleavage reaction. Two recent studies have focused on kinetic parameters of hammerhead ribozyme mediated tran.~cleavage of RNA substrates. Goodchild and Kohli [22 o] examined the effects of the length of base-paired flanking sequences and of the internal stem-loop structure on ribozyme kinetics. They observed that reducing the length of flanking sequences from 20 to 12 base pairs increased the rate of cleavage 10-fold at 37°C. Interestingly, they also observed that deletion of the internal stem-loop also markedly improved initial rate kinetics. Taylor and Rossi [23"] examined the effects of non-base-paired flanking sequences on hammerhead ribozyme catalysis. When 75 non-basepaired, unstructured, flanking sequences were appended to the base-paired portions of the ribozyme, there were minimal kinetic consequences. In contrast, when the ribozyme was embedded in 153 bases of a highly structured RNA, the turnover efficiency was reduced approximately eight-fold. The results from both of these studies provide important information for evaluating the design of ribozyme motifs for intracellular expression.
In vivo or intracellular applications of
ribozymes There have been several reports of hammerhead ribozyme mediated inhibition of target RNA function [24-26], but none of these studies has provided direct evidence that the ribozyme was cleaving its target as predicted. The first direct demonstration that a wan.s-acting fibozyme can cleave a target RNA in vivo came from experiments by Saxena and Ackerman [27 °] in which hammerhead ribozymes targeted to an exposed loop of 28S rRNA were injected into Xenopus oocytes. Targeted cleavage of the 28S rRNA substrate, as well as destruction of the translational capacity of the affected ribosomes were demonstrated. A very elegant set of experiments by Sioud and Drlica [28-.] demonstrating ribozymemediated cleavage of a targeted RNA in vivo used an inducible expression system for both ribozyrne and substrate in the bacterium E coli. In these studies a hammerhead ribozyme was designed to cleave the human immunodeficiency virus-1 integrase RNA expressed in E co//. The results of these experiments provide direct demonstrations of ribozyme-mediated reduction of the levels of integrase RNA and translated product and clearly demonstrate that the hammerhead catalytic center embedded in the anti-sense RNA transcript is necessary for this to occur. These studies are also important for demonstrating the usefulness of a simple biological system such as E. coli to model effective tran.~acting ribozymes. The use of a ribozyme to alter the state of a transformed cell has recently been demonstrated by Scan-
6
Analytical biotechnolosy lon et al. [29], Cells resistant to the chemotherapeutic agent c~platin exhibit elevated levels of several RNAs and proteins, including that of the important transcriptional factor c-fos In these studies, a hammerhead ribozyme targeted to the c-fos mRNA was placed under transcriptional comrol of the mouse mammary tumor virus glucocorticoid-responsive promoter. It was demonstrated that, upon induction of the ribozyme, cancerous cells resistant to c~platin become sensitive to this agent as a partial consequence of the ensuing reduction in the c-fos mRNA levels. This reduction in c-fos is accompariled by a cascade reduction of several other fo~responsire mRNAs encoding enzymes involved in DNA repair. A mutant form of the ribozyme with reduced cleavage activity had a marginal effect in restoring sensitivity to c/s-platin, and a ribozyme in the anti-sense or wrong offentation had no effect. As well as confirming the direct involvemem of c-los in the expression of several enzymes involved in DNA repair, these experiments point to the potential possibility of using fibozymes in conjunction with chemotherapeutic agents.
Conclusions The discovery of enzymatic activities associated with RNA molecules has fueled a great amount of research to determine the mechanism(s) of ribozyme-mediated cleavage (and ligation) reactions, as well as to find ways of putting the cleavage capabilities of ribozymes to use in enzymatic-antisense reagents. At this time, the two most well understood mechanisms are those of the Tetrahymena intervening sequence ribozyme and the plant viroid(and virusoid)-derived hammerhead catalytic motif. Within the next year we should witness significant progress in the elucidation of the catalytic mechanisms of the hepatitis delta virus and hairpin catalytic reactions, which at this time are very poorly understood. One of the major goals of ribozyme research is to put these important molecules to work as enzymatic antisense agents. Because of their great specificity in targeting substmtes and the fact that, unlike antisense DNA, they can be synthesized endogenously within a cell, ribozymes hold great promise for inactivating any RNA target of choice. Unlike proteins, for which we do not dearly understand the rules for folding and substmte specificity, ribozymes targeted to any RNA target can be rapidly designed and tested. The major limitations in the use of ribozymes will be interaction with the target RNA as these involve complementary base pairing. Intramolecular structures of the target RNAs, as well as protein interactions with the RNA make efficient interactions unlikely for many RNAs. Understanding how to maximize ribozyme-substrate interactions in a living cell is the next major challenge in this field.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ** of outstanding interest I.
2.
3.
KRUGERK, GRABOWSKIPJ, ZAUGAJ, SANDSJ, Go'rrSCHLtNG DE, CECH TPC Self-splicing RNA: Autoexcision and Autocyclization of the Ribosomal RNA Intervening Sequence of Tetrahymena~ Cell 1982, 31:147-157. GUERRIER-TAKADAC, GARDINERK, MARSH R, PACE N, ALTMAN S: Catalytic Activity of an RNA Molecule Prepared by Transcription In Vitr~ Cell 1983, 35:849--857. HERSCHLAGD, CECH TPC DNA Cleavage Catalyzed by the Ribozyme from Tetrahymena~ Nature 1990, 344:405--409.
ROBERTSONDL, JOYCE GF: Selection in vitro of an lENA Enzymc that Specifically Cleaves Single-stranded DNA. Nature 1990, 344:467-468. Describes an in vitro enrichment system using the Tetrabymena core ribozyme in conjunction with the polymerase chain reaction to enrich for variants of the ribozyme that can cleave and ligate DNA. 4. ,.
5. ~he
FORSTEIiAC, ALTMAN S: External Guide Sequences for an RNA Enzyme. Science 1990, 249:783-786. substrate requirements for/~ co/i RNAse P are demonstrated. On the basis of the minimum required structure of RNAse P necessary for cleavage, a model is proposed for forming external guide sequences with any target RNA to make that RNA a substrate for RNAse P. The external guide sequence is provtded in trar~ and is capable of com. plementary base pairing with the target or substrate RNA. It is necessary to have an external guide sequence to form a substrate for recognition and cleavage by the RNAse P holoenzyme. 6.
FORSTERAC, SYMONS RH: Self-cleavage of Plus and Minus RNAs of a VLrusoid and a Structural Model for the Active Sites. Cell 1987, 49:211-220.
7.
HAMPEL A, TRrl-ZPc RNA Catalytic Properties of the Minimum ( - ) sTRSV Sequence. Biochemistry 1989, 28:4929-4933.
8.
UHLENBECKOC: A Small Catalytic Oligoribonucleotide. Nature 1987, 328:596-600. HASELOFFJ, GERLACHWL Simple Enzymes with New and Highly Specific Endoribonuclease Activity. Nature 1988, 334:585-591 SHARMEENL KUO M, DINTER-GOTIZIEBG, TAYLORJ: Antigenomic RNA of Human Hepatitis Delta Virus can Undergo Self-cleavage. J Virol 1988, 62:2674-2679. BELINSKYMG, DINTER-GOTI'L1EBG: Non-ril~zyrne Sequences Enhance Self-cleavage of Ribozymes Derived from Hepatitis Delta Virus. Nucleic Acids Res 1991, 19:559-564. PERRO'ITAAT, BEEN MD: The Self-cleaving Domain from the Genomic RNA of Hepatitis Delta Virus: Sequence Requiremerits and the Effects of Denaturant. Nucleic Acids Res 1990, 18:6821-6827.
9.
10.
11
12.
13.
14. **
PERROTrAAT, BEEN MD: A Pseudoknot.like Structure Required for Efficient Self-cleavage of Hepatitis Delta Virus RN& Nature 1991, 350:434--436.
GREENR, ELLINGTONAD, SZOSTAKJ'~7: In vitro Genetic Analysis of the Tetrahymena Self-splicing intron. Nature 1991, 347:406-408. This paper describes an in vitro system for the rapid evolution and enrichment of structural variants of Tetrabymena.type ribozymes with enhanced catalytic activity or altered substrate recognition features. This clever technique is very similar to that used by Robertson and Joyce [4"'1. With this technique, it should be possible to select and enrich for variant ribozymes that can cleave any desired RNA (or single.stranded DNA) target.
K i b o z y m e s Rossi 15.
BERZAL-HERRANZA, JOSEPH S, BURKEJM: I n Vitro selection of Active Hairpin Ribozymes by sequential RNA-catalyzed Cleavage and Ligation Reactions. Gene Dev 1992, 6:129-134.
16.
HERSCHLAGD, CECIl TE Catalysis of RNA Cleavage by the Tet r a h y m e n a t b e r m o p b t l a Ribozyme. 2. Kinetic Description of the Reaction of an RNA Substrate that Forms a Mismatch at the Active Site. Bt~/aem~ry 1990, 29:10172-10180.
17. ••
PYLE AM, CECH TR: Ribozyme Recognition of RNA by Tertiary Interactions with Specific Ribose 2'OH groups. N a t u r e 1991, 350:628--631. A shortened form of the Tetrahymena intron ribozyme catalyzes sequence-specific cleavage of substrate RNAs. The association of RNA substrates with the ribozyme internal guide sequence exhibits a binding energy much greater than that predicted from base-pairing alone. By comparing equilibrium binding constants of substrates harboring RNA or DNA at critical positions in the substrate, they demonstrate that this increased binding energy is caused by a specific interaction of a 2' hydroxyl in the substrate with an undefined site in the intron. These studies demonstrate that tertiary interactions can play a key role in stabilizing duplexed RNAs. 18. **
RUFFNERDE, ST•RiO GD, UHLENBECKOC: Soquence Requirements of the Hammerhead RNA self-cleavage Reaction. Bit> chem/stry 1990, 29:10695-10702. This is an exhaustive genetic and kinetic analysis of the hammerhead ribozyme, and should serve as a key reference for anyone designing a tran.~acting hammerhead ribozyme. Most importantly, the relative rates of cleavage of various XLVX(where X is any base) cleavage sites are determined. 19
PERREAULT J-P, LABUDAD, USMANN, YANGJ-H, CEDERGRENR: Relationship Between 2' Hydroxyls and Magnesium BInding In the Hammerhead RNA Domain: A Model for Ribozyme Catalysis. B/ochem/stry 1991, 30:4020-4025.
20.
SLIM G, GAIT MJ: ConligurationaUy Defined Phosphorothioate-containing Ofigoribonucleotides in the Study of the Mechanism of Cleavage of Hammerhead Ribozymes. Nucle~ Acids Res 1991, 19:1183-1188.
21
PIEKENWA, OLSEN DB, BENSELERF, AURUP H, ECKSTEIN F: Kinetic Characterization of Ribonuclease 2' Modified Hammerhead Rlbozymes. Science 1991, 253:314-317.
22. •
G(X)DCmLDJ, KOHI,I V: Ribozymes that Cleave an RNA sequence from Human Immunodeficiency Virus: the Effect of Flanking Sequence on Rate. Arch Bic~hem B i ( ~ 1991, 284:386-391. A very nice systematic analysis of the effects of varying basepairing sequence length on catalytic turnover of a hammerhead ribozyme using an HIV-1 substrate at 37°C.
23. •
TAYLORN, ROSSlJJ: Ribozyme-mediated Cleavage of an HIV-1 gag RNA: The Effects of Non-targeted Sequences and Secondary Structure of Ribozyme Cleavage Activity. Ant/sense Res Dev 1991, 1:173-186. This is the first critical examination of the potential effects of non-base pairing sequences on catalytic activity of hammerhead ribozymes. This paper also describes a system for the production of hammerhead type ribozymes in E. coll. 24.
CAMERONFH, JENNINGS PA. Specific Gene Suppression by Engineered Ribozymes in Monkey Cells. Proc Natl Acad Sci USA 1989, 86:9139--9143.
25.
COTI'ENM, BIRNSTIELML: Ribozyme Mediated Destruction of RNA in v i v a EMBO J 1989, 8:3861-3866.
26.
SARVERN, CANTIN E, CHANG P, LADNE P, STEPHENS D, ZAIA J, ROSS)JJ: Rtbozymes as Potential Anti-HIV-1 Therapeutic Agents. Science 1990, 247:1222-1225.
27. •
SAXENASK, ACKERMANEJ: Ribozymes Correctly Cleave a Model Substrate and Endogenous RNA tn v i v a J Biol Chem 1990, 265:17106-17109. This is the first demonstration of cleavage mediated by a tran,~acting ribozyme in viva The data presented also point out the importance of choosing an accessible target for cleavage. 28. 00
SIOUDM, DRUCAK: Prevention of Human Immunodeficiency Virus Type 1 Integrase Expression in E$chertchta ¢olt by a Ribozyme. Proc N a a A c a d Sci USA 1991, 88:7303-7307. This carefully performed study used a hammerhead ribozyme targeted to HIV-1 integrase RNA that was engineered to be expressed in E. co//. When the ribozyme was expressed from an inducible promoter, it destroyed the target RNA sufficiently to result in complete blockage of protein expression. Some cleavage products were detected by northern gel analysis from ribozyme-expressing constructs. Because of the short half-lives of RNAsin E co/i, this system can be considered a stringent test of ribozyme function. The results from this study, therefore, strongly support the contention that ribozymes are more effective than standard antisense RNA at blocking gene expression. This study also demonstrates the usefulneSS of exploiting E. coliin screening for active ribozyme variants. 29.
SCANLONKJ, JIaO L FUNATO T, WANG W, TONE T, ROSSl JJ, KASHANI-SABETM: Ribozyme-mediated Cleavage of c-fos mRNA Reduces Gene Expression of DNA Synthesis Enzymes and Metallothionein. Proc Natl Acad Sci USA 1991, 88:10591-10595.
JJ Rossi, Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
7
Introduction RNA, besides being an informational molecule, can also possess enzymatic properties. Those RNAs having this property have been termed ribozymes. Since the initial discoveries of enzymatic RNAs by Cech and Altman and their colleagues [1,2], there have been numerous descriptions of ribozymes found throughout nature. Aside from their important roles in biological processes such as splicing and RNA modification, as well as the implications for macromolecular evolution, the fact that RNAs can be enzymatic has fostered a whole new technology of transacting, catalytic, antisense ribozymes. This new class of antisense agents allows specific targeting of any desired RNA, followed by cleavage and functional inactivation of that target. Certain of these molecules have truly catalytic properties in that they are not consumed or modified in the reaction, and can turn over multiple substrates. In this review I will discuss first the various catalytic motifs, then the past year's literature describing structural and kinetic analyses of ribozymes and, finally, the most recent papers describing the applications of ribozymes in vivo as catalytic antisense agents.
RNA catalytic motifs At present, there are four RNA catalytic motifs that are potentially useful for tran.~mediated cleavage of target RNAs (Fig. 1). Perhaps the most thoroughly studied is that of the self-splicing intervening sequence of Tetrah)~ mena thermophila, first characterized by Cech and colleagues [ 1 ]. Although this RNA enzyme possesses a rather complex structure, it has been engineered to cleave a variety of substrates, even including single-stranded DNA [3,4"]. RNAse P is a ubiquitous enzyme that processes the 5' leader sequences from pre-transfer RNAs and has an RNA component that has been shown to be the catalytic entity for the bacterial form of the enzyme [2]. Forster and Altman [5"] have characterized the substrate requirements for the E. coli enzyme, and have demonstrated that it can cleave a non-tRNA duplex provided
that the target is in a helix and that the antisense to the target RNA has a CCA sequence at its 3' terminus (Fig. lb). Thus, in principle, any RNA can be targeted as a substrate for RNAse P provided that the CCA-containing antisense RNA can be annealed to the target RNA. Two RNA catalytic motifs that originate from plant viroids and virusoids have received a great deal of attention for their use in antisense ribozymes. These are the 'hammerhead' [6] and 'hairpin' [7] cleavage domains, illustrated in Fig. 1 (c and d). Uhlenbeck [8] and Haseloff and Gerlach [9] demonstrated that the hammerhead catalytic motif could be incorporated into trans-acting antisense RNAs, thereby imparting enzymatic activity on these molecules. The hairpin catalytic domain was subsequently shown to be useful for incorporation into tran.~acting ribozymes as well [6]. One other ribozyme catalytic domain that has potential for inclusion into trans.acting ribozymes is that derived from the human hepatitis delta virus. The catalytic center of this ribozyme, like that of the self-splicing introns and RNAse P, is more complex than that of the hammerhead and is heavily dependent upon structure for its activity [10-12]. A number of investigators have recently reported their efforts to define the catalytic domain of this molecule [11-13]. Once this ribozyme is better defined, it should also find application for inclusion into catalytic antisense RNAs.
Structural and kinetic analyses of ribozyme-mediated catalysis A large number of studies have sought to provide a better understanding of the mechanism of ribozyme-mediated catalysis, as well as to provide an intelligent framework for designing more active ribozymes. Two groups have used an in vitro evolution approach to create Tetrahymena-type ribozymes with altered or enhanced catalytic properties [4.-,14-.]. These investigators took advantage of the fact that the Tetrahymena ribozyme has both cleavage and ligation activities, and set up in vitro systems for enriching and amplifying ribozyme variants
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with desired characteristics. In these studies, a randomized population of modified ribozymes was incubated with the desired, specific substrate molecule that is to be cleaved. Concommitant with the cleavage event, substrate sequences lying 3' to the site of cleavage become ligated to ~ e intron. The intron, with its covalently attached cleavage product is amplified by the polymerase chain reaction using primers complementary to the cleavage product and the 5' end of the intron. At least one of the primers contains the sequence specifying transcription from a bacteriophage promoter such as T7. The amplified DNA is transcribed, and the whole process repeated several times until the modified form of the ribozyme that is most active in cleaving the desired substrate is the major
I
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Fig. 1. Four different RNA catalytic motifs: (a) Tetrahymena self-splicing intervening sequence ribozyme; (b) Esch erichia coil RNAse P, comprising RNAse P proteins and M1 RNA to make up the holoenzyme; (c) hammerhead ribozyme; and (d) the tobacco ringspot virus (negative strand) hairpin ribozyme. The guide sequence for the Tetrahymena ribozyme is IGS for internal guide sequence, and the RNAse P guide sequence is EGS for external guide sequence. The IGS is part of the Tetrahymena core ribozyme and is its primary binding site for substrate. Specific complementary base-pairing interactions between this site and the substrate confer specificity to the ribozyme reaction. The EGS is an 'antisense' RNA with an appended ACCA at the 3' end, which is provided in trans. Complementary base pairing of the EGS with the target RNA results in a duplex substrate for RNAse P, by mimicking the CCA stem of the natural tRNA substrate molecules. The symbols N and N' denote no specificity for nucleotides at these positions, and roman numerals indicate regions required for ribozyme functions. All four ribozymes are depicted as they would interact with substrates 'in trans'. The substrates and the sites of cleavage are indicated.
amplified product. By using DNA substrates in place of RNA, Robertson and Joyce [4 °°] were able to generate variants that have single-stranded DNA cleavage capabilities. Using a somewhat different strategy, Green et at [14o*] began with a pool of mutant ribozymes in which the wild-type sequence was present at an extremely low level. By carrying out only three cycles of selection and amplification for enhanced ribozyme function, they were able to isolate the wild-type ribozyme as well as structural variants as active as the wild type. Using such m vitro selection schemes, it should be possible to isolate variant Tetrahymena-type ribozymes capable of acting on virtually any substrate of choice. Unfortunately, it has not yet been possible to apply such a directed evolution strategy
Ribozymes Rossi S to the hammerhead-type ribozymes. Recently, an in vitro selection scheme, similar in principal to those used for the Terahymena ribozyme intron, has been developed for the hairpin ribozyme, allowing selection for cleavage of novel substrates [15]. Cech and his colleagues [16,17.-] have used the Tetrah?~ mena ribozyme system to determine the general principles of ribozyme-substrate interactions. A particularly interesting observation involves a tertiary interaction between a 2' hydroxyl group at a particular position in the susbtrate with residues within the Tetrahymena intron ribozyme. This single interaction results in an increase in binding energy of 4.1 kcal m o l - 1 over that expected for simple RNA-RNA base pairing [17-.]. This increased binding energy greatly reduces the efficiency of product dissociation, thereby slowing the turnover rate of the ribozyme. Such tertiary interactions probably exist for other types of ribozyme-substrate interactions as well and it may thus be possible to increase catalytic turnover of ribozymes by altering or changing these interactions. Several key papers analyzing hammerhead-type ribozymes have helped to define the essential nucleotides and magnesium-binding sites within the catalytic center. Ruffner et al. [18..] have rigorously evaluated the contribution to catalytic efficiency of every nucleotide in the hammerhead structure. Their data clearly demonstrate that the catalytic efficiency can be grossly altered by several different single base substitutions within the highly conserved hammerhead domain. The choice of cleavage site is also an important consideration, with GUC being the kinetically most favorable site. Perreault et al. [19] substituted deoxyribonucleotides at various positions in the hammerhead structure to assess the role of the 2' hydroxyl group on the ribose moiety in the binding of magnesium cations. Three positions, two of which reside in the hammerhead and one that is adjacent to the site of cleavage, were found to be critical for magnesium binding. On the basis of these observations, a molecular model for hammerhead ribozyme mediated cleavage is presented, which involves magnesium binding at these three critical positions and at the scissile phosphate. Slim and Gait [20] substituted configurationally defined phosphorothioate-containing oligoribonucleotides at a single site in a hammerhead ribozyme cleavage site to investigate the importance of magnesium ion binding to the pro-R oxygen of the cleaved phosphodiester. Their findings provide further evidence that a magnesium ion is bound to the pro-R oxygen in the transition state of the cleavage reaction. In combination, these results support a model in which the role of the magnesium ion is primarily catalytic rather than structural. Pieken et aL [21] chemically synthesized hammerhead ribozymes with either 2' fluoro or 2' aminonucleotides. The incorporation of 2' fluorouridines, 2' fluorocytidines or 2' aminouridines did not appreciably decrease catalytic efficiency, but did markedly increase the resistance of these ribozymes to ribonuclease degradation, an important consideration for exogenous delivery of ribozymes to cells in culture. Each of the above studies has provided greater insight into the roles of the highly con-
served bases and ribose moieties in the catalytic center of the hammerhead ribozyme and at the site of cleavage of its substrates. Further physical studies, however, using nuclear magnetic resonance spectroscopy and X-ray crystallographic analyses will be necessary for a full understanding of the mechanism of the site-specific cleavage reaction. Two recent studies have focused on kinetic parameters of hammerhead ribozyme mediated tran.~cleavage of RNA substrates. Goodchild and Kohli [22 o] examined the effects of the length of base-paired flanking sequences and of the internal stem-loop structure on ribozyme kinetics. They observed that reducing the length of flanking sequences from 20 to 12 base pairs increased the rate of cleavage 10-fold at 37°C. Interestingly, they also observed that deletion of the internal stem-loop also markedly improved initial rate kinetics. Taylor and Rossi [23"] examined the effects of non-base-paired flanking sequences on hammerhead ribozyme catalysis. When 75 non-basepaired, unstructured, flanking sequences were appended to the base-paired portions of the ribozyme, there were minimal kinetic consequences. In contrast, when the ribozyme was embedded in 153 bases of a highly structured RNA, the turnover efficiency was reduced approximately eight-fold. The results from both of these studies provide important information for evaluating the design of ribozyme motifs for intracellular expression.
In vivo or intracellular applications of
ribozymes There have been several reports of hammerhead ribozyme mediated inhibition of target RNA function [24-26], but none of these studies has provided direct evidence that the ribozyme was cleaving its target as predicted. The first direct demonstration that a wan.s-acting fibozyme can cleave a target RNA in vivo came from experiments by Saxena and Ackerman [27 °] in which hammerhead ribozymes targeted to an exposed loop of 28S rRNA were injected into Xenopus oocytes. Targeted cleavage of the 28S rRNA substrate, as well as destruction of the translational capacity of the affected ribosomes were demonstrated. A very elegant set of experiments by Sioud and Drlica [28-.] demonstrating ribozymemediated cleavage of a targeted RNA in vivo used an inducible expression system for both ribozyrne and substrate in the bacterium E coli. In these studies a hammerhead ribozyme was designed to cleave the human immunodeficiency virus-1 integrase RNA expressed in E co//. The results of these experiments provide direct demonstrations of ribozyme-mediated reduction of the levels of integrase RNA and translated product and clearly demonstrate that the hammerhead catalytic center embedded in the anti-sense RNA transcript is necessary for this to occur. These studies are also important for demonstrating the usefulness of a simple biological system such as E. coli to model effective tran.~acting ribozymes. The use of a ribozyme to alter the state of a transformed cell has recently been demonstrated by Scan-
6
Analytical biotechnolosy lon et al. [29], Cells resistant to the chemotherapeutic agent c~platin exhibit elevated levels of several RNAs and proteins, including that of the important transcriptional factor c-fos In these studies, a hammerhead ribozyme targeted to the c-fos mRNA was placed under transcriptional comrol of the mouse mammary tumor virus glucocorticoid-responsive promoter. It was demonstrated that, upon induction of the ribozyme, cancerous cells resistant to c~platin become sensitive to this agent as a partial consequence of the ensuing reduction in the c-fos mRNA levels. This reduction in c-fos is accompariled by a cascade reduction of several other fo~responsire mRNAs encoding enzymes involved in DNA repair. A mutant form of the ribozyme with reduced cleavage activity had a marginal effect in restoring sensitivity to c/s-platin, and a ribozyme in the anti-sense or wrong offentation had no effect. As well as confirming the direct involvemem of c-los in the expression of several enzymes involved in DNA repair, these experiments point to the potential possibility of using fibozymes in conjunction with chemotherapeutic agents.
Conclusions The discovery of enzymatic activities associated with RNA molecules has fueled a great amount of research to determine the mechanism(s) of ribozyme-mediated cleavage (and ligation) reactions, as well as to find ways of putting the cleavage capabilities of ribozymes to use in enzymatic-antisense reagents. At this time, the two most well understood mechanisms are those of the Tetrahymena intervening sequence ribozyme and the plant viroid(and virusoid)-derived hammerhead catalytic motif. Within the next year we should witness significant progress in the elucidation of the catalytic mechanisms of the hepatitis delta virus and hairpin catalytic reactions, which at this time are very poorly understood. One of the major goals of ribozyme research is to put these important molecules to work as enzymatic antisense agents. Because of their great specificity in targeting substmtes and the fact that, unlike antisense DNA, they can be synthesized endogenously within a cell, ribozymes hold great promise for inactivating any RNA target of choice. Unlike proteins, for which we do not dearly understand the rules for folding and substmte specificity, ribozymes targeted to any RNA target can be rapidly designed and tested. The major limitations in the use of ribozymes will be interaction with the target RNA as these involve complementary base pairing. Intramolecular structures of the target RNAs, as well as protein interactions with the RNA make efficient interactions unlikely for many RNAs. Understanding how to maximize ribozyme-substrate interactions in a living cell is the next major challenge in this field.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ** of outstanding interest I.
2.
3.
KRUGERK, GRABOWSKIPJ, ZAUGAJ, SANDSJ, Go'rrSCHLtNG DE, CECH TPC Self-splicing RNA: Autoexcision and Autocyclization of the Ribosomal RNA Intervening Sequence of Tetrahymena~ Cell 1982, 31:147-157. GUERRIER-TAKADAC, GARDINERK, MARSH R, PACE N, ALTMAN S: Catalytic Activity of an RNA Molecule Prepared by Transcription In Vitr~ Cell 1983, 35:849--857. HERSCHLAGD, CECH TPC DNA Cleavage Catalyzed by the Ribozyme from Tetrahymena~ Nature 1990, 344:405--409.
ROBERTSONDL, JOYCE GF: Selection in vitro of an lENA Enzymc that Specifically Cleaves Single-stranded DNA. Nature 1990, 344:467-468. Describes an in vitro enrichment system using the Tetrabymena core ribozyme in conjunction with the polymerase chain reaction to enrich for variants of the ribozyme that can cleave and ligate DNA. 4. ,.
5. ~he
FORSTEIiAC, ALTMAN S: External Guide Sequences for an RNA Enzyme. Science 1990, 249:783-786. substrate requirements for/~ co/i RNAse P are demonstrated. On the basis of the minimum required structure of RNAse P necessary for cleavage, a model is proposed for forming external guide sequences with any target RNA to make that RNA a substrate for RNAse P. The external guide sequence is provtded in trar~ and is capable of com. plementary base pairing with the target or substrate RNA. It is necessary to have an external guide sequence to form a substrate for recognition and cleavage by the RNAse P holoenzyme. 6.
FORSTERAC, SYMONS RH: Self-cleavage of Plus and Minus RNAs of a VLrusoid and a Structural Model for the Active Sites. Cell 1987, 49:211-220.
7.
HAMPEL A, TRrl-ZPc RNA Catalytic Properties of the Minimum ( - ) sTRSV Sequence. Biochemistry 1989, 28:4929-4933.
8.
UHLENBECKOC: A Small Catalytic Oligoribonucleotide. Nature 1987, 328:596-600. HASELOFFJ, GERLACHWL Simple Enzymes with New and Highly Specific Endoribonuclease Activity. Nature 1988, 334:585-591 SHARMEENL KUO M, DINTER-GOTIZIEBG, TAYLORJ: Antigenomic RNA of Human Hepatitis Delta Virus can Undergo Self-cleavage. J Virol 1988, 62:2674-2679. BELINSKYMG, DINTER-GOTI'L1EBG: Non-ril~zyrne Sequences Enhance Self-cleavage of Ribozymes Derived from Hepatitis Delta Virus. Nucleic Acids Res 1991, 19:559-564. PERRO'ITAAT, BEEN MD: The Self-cleaving Domain from the Genomic RNA of Hepatitis Delta Virus: Sequence Requiremerits and the Effects of Denaturant. Nucleic Acids Res 1990, 18:6821-6827.
9.
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14. **
PERROTrAAT, BEEN MD: A Pseudoknot.like Structure Required for Efficient Self-cleavage of Hepatitis Delta Virus RN& Nature 1991, 350:434--436.
GREENR, ELLINGTONAD, SZOSTAKJ'~7: In vitro Genetic Analysis of the Tetrahymena Self-splicing intron. Nature 1991, 347:406-408. This paper describes an in vitro system for the rapid evolution and enrichment of structural variants of Tetrabymena.type ribozymes with enhanced catalytic activity or altered substrate recognition features. This clever technique is very similar to that used by Robertson and Joyce [4"'1. With this technique, it should be possible to select and enrich for variant ribozymes that can cleave any desired RNA (or single.stranded DNA) target.
K i b o z y m e s Rossi 15.
BERZAL-HERRANZA, JOSEPH S, BURKEJM: I n Vitro selection of Active Hairpin Ribozymes by sequential RNA-catalyzed Cleavage and Ligation Reactions. Gene Dev 1992, 6:129-134.
16.
HERSCHLAGD, CECIl TE Catalysis of RNA Cleavage by the Tet r a h y m e n a t b e r m o p b t l a Ribozyme. 2. Kinetic Description of the Reaction of an RNA Substrate that Forms a Mismatch at the Active Site. Bt~/aem~ry 1990, 29:10172-10180.
17. ••
PYLE AM, CECH TR: Ribozyme Recognition of RNA by Tertiary Interactions with Specific Ribose 2'OH groups. N a t u r e 1991, 350:628--631. A shortened form of the Tetrahymena intron ribozyme catalyzes sequence-specific cleavage of substrate RNAs. The association of RNA substrates with the ribozyme internal guide sequence exhibits a binding energy much greater than that predicted from base-pairing alone. By comparing equilibrium binding constants of substrates harboring RNA or DNA at critical positions in the substrate, they demonstrate that this increased binding energy is caused by a specific interaction of a 2' hydroxyl in the substrate with an undefined site in the intron. These studies demonstrate that tertiary interactions can play a key role in stabilizing duplexed RNAs. 18. **
RUFFNERDE, ST•RiO GD, UHLENBECKOC: Soquence Requirements of the Hammerhead RNA self-cleavage Reaction. Bit> chem/stry 1990, 29:10695-10702. This is an exhaustive genetic and kinetic analysis of the hammerhead ribozyme, and should serve as a key reference for anyone designing a tran.~acting hammerhead ribozyme. Most importantly, the relative rates of cleavage of various XLVX(where X is any base) cleavage sites are determined. 19
PERREAULT J-P, LABUDAD, USMANN, YANGJ-H, CEDERGRENR: Relationship Between 2' Hydroxyls and Magnesium BInding In the Hammerhead RNA Domain: A Model for Ribozyme Catalysis. B/ochem/stry 1991, 30:4020-4025.
20.
SLIM G, GAIT MJ: ConligurationaUy Defined Phosphorothioate-containing Ofigoribonucleotides in the Study of the Mechanism of Cleavage of Hammerhead Ribozymes. Nucle~ Acids Res 1991, 19:1183-1188.
21
PIEKENWA, OLSEN DB, BENSELERF, AURUP H, ECKSTEIN F: Kinetic Characterization of Ribonuclease 2' Modified Hammerhead Rlbozymes. Science 1991, 253:314-317.
22. •
G(X)DCmLDJ, KOHI,I V: Ribozymes that Cleave an RNA sequence from Human Immunodeficiency Virus: the Effect of Flanking Sequence on Rate. Arch Bic~hem B i ( ~ 1991, 284:386-391. A very nice systematic analysis of the effects of varying basepairing sequence length on catalytic turnover of a hammerhead ribozyme using an HIV-1 substrate at 37°C.
23. •
TAYLORN, ROSSlJJ: Ribozyme-mediated Cleavage of an HIV-1 gag RNA: The Effects of Non-targeted Sequences and Secondary Structure of Ribozyme Cleavage Activity. Ant/sense Res Dev 1991, 1:173-186. This is the first critical examination of the potential effects of non-base pairing sequences on catalytic activity of hammerhead ribozymes. This paper also describes a system for the production of hammerhead type ribozymes in E. coll. 24.
CAMERONFH, JENNINGS PA. Specific Gene Suppression by Engineered Ribozymes in Monkey Cells. Proc Natl Acad Sci USA 1989, 86:9139--9143.
25.
COTI'ENM, BIRNSTIELML: Ribozyme Mediated Destruction of RNA in v i v a EMBO J 1989, 8:3861-3866.
26.
SARVERN, CANTIN E, CHANG P, LADNE P, STEPHENS D, ZAIA J, ROSS)JJ: Rtbozymes as Potential Anti-HIV-1 Therapeutic Agents. Science 1990, 247:1222-1225.
27. •
SAXENASK, ACKERMANEJ: Ribozymes Correctly Cleave a Model Substrate and Endogenous RNA tn v i v a J Biol Chem 1990, 265:17106-17109. This is the first demonstration of cleavage mediated by a tran,~acting ribozyme in viva The data presented also point out the importance of choosing an accessible target for cleavage. 28. 00
SIOUDM, DRUCAK: Prevention of Human Immunodeficiency Virus Type 1 Integrase Expression in E$chertchta ¢olt by a Ribozyme. Proc N a a A c a d Sci USA 1991, 88:7303-7307. This carefully performed study used a hammerhead ribozyme targeted to HIV-1 integrase RNA that was engineered to be expressed in E. co//. When the ribozyme was expressed from an inducible promoter, it destroyed the target RNA sufficiently to result in complete blockage of protein expression. Some cleavage products were detected by northern gel analysis from ribozyme-expressing constructs. Because of the short half-lives of RNAsin E co/i, this system can be considered a stringent test of ribozyme function. The results from this study, therefore, strongly support the contention that ribozymes are more effective than standard antisense RNA at blocking gene expression. This study also demonstrates the usefulneSS of exploiting E. coliin screening for active ribozyme variants. 29.
SCANLONKJ, JIaO L FUNATO T, WANG W, TONE T, ROSSl JJ, KASHANI-SABETM: Ribozyme-mediated Cleavage of c-fos mRNA Reduces Gene Expression of DNA Synthesis Enzymes and Metallothionein. Proc Natl Acad Sci USA 1991, 88:10591-10595.
JJ Rossi, Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
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